Genome-wide identification and expression analysis of the U-box E3 ubiquitin ligase gene family related to bacterial wilt resistance in tobacco (Nicotiana tabacum L.) and eggplant (Solanum melongena L.)

Abstract

The E3 enzyme in the UPS pathway is a crucial factor for inhibiting substrate specificity. In Solanaceae, the U-box E3 ubiquitin ligase has a complex relationship with plant growth and development, and plays a pivotal role in responding to various biotic and abiotic stresses. The analysis of the U-box gene family in Solanaceae and its expression profile under different stresses holds significant implications. A total of 116 tobacco NtU-boxs and 56 eggplant SmU-boxs were identified based on their respective genome sequences. Phylogenetic analysis of U-box genes in tobacco, eggplant, tomato, Arabidopsis, pepper, and potato revealed five distinct subgroups (I-V). Gene structure and protein motifs analysis found a high degree of conservation in both exon/intron organization and protein motifs among tobacco and eggplant U-box genes especially the members within the same subfamily. A total of 15 pairs of segmental duplication and 1 gene pair of tandem duplication were identified in tobacco based on the analysis of gene duplication events, while 10 pairs of segmental duplication in eggplant. It is speculated that segmental duplication events are the primary driver for the expansion of the U-box gene family in both tobacco and eggplant. The promoters of NtU-box and SmU-box genes contained cis-regulatory elements associated with cellular development, phytohormones, environment stress, and photoresponsive elements. Transcriptomic data analysis shows that the expression levels of the tobacco and eggplant U-box genes in different tissues and various abiotic stress conditions. Using cultivar Hongda of tobacco and cultivar Yanzhi of eggplant as materials, qRT-PCR analysis has revealed that 15 selected NtU-box genes and 8 SmU-box may play important roles in response to pathogen Ras invasion both in tobacco and eggplant.

Keywords: Nicotiana tabacum L.; Solanum melongena L.; U-box; biotic stress; expression analysis; phylogenetic analysis.

Figure 1

Gene distribution and duplication (A) NtU-box genes on the 24 chromosomes in tobacco (B) SmU-box genes on the 12 chromosomes in eggplant. Tandem-duplicated gene pairs of Nt19 are marked with red box, and segmental duplication genes are connected by red line.

Figure 2

Collinearity analyses of U-box genes among tobacco, eggplant tomato and Arabidopsis. (A) Collinearity analyses among Arabidopsis thaliana, Nicotiana tabacum and Solanum melongena. (B) Collinearity analyses among Solanum Lycopersicon, Nicotiana tabacum and Solanum melongena. The gray line represents the co-collinearity of all genes among the three species, and the purple and blue line represents the collinearity among members of the U-box gene family.

Figure 3

Gene structure and evolution of U-box family in tobacco (A) Phylogenetic relationships of NtU-boxs. Different subgroups were marked with different colors. (B) Intron-exon structure of NtU-boxs. UTR and CDS are represented by different colors. The scale bar of bottom demonstrates the length of exons and introns.

Figure 4

Gene structure and evolution of U-box family in eggplant. (A) Phylogenetic relationships of SmU-boxs. Different subgroups were marked with different colors. (B) Intron-exon structure of SmU-boxs. UTR and CDS are represented by different colors. The scale bar of bottom demonstrates the length of exons and introns.

Figure 5

Conserved motifs for U-box proteins in tobacco. Different motifs are showed with different colored boxes and numbers (1-20).

Figure 6

Conserved motifs for U-box proteins in eggplant. Different motifs are showed with different colored boxes and numbers (1-20).

Figure 7

Phylogenetic relationship of 116 NtU-box and 56 SmU-box proteins, along with another 256 published U-box proteins. The phylogenetic relationships were generated by using MEGA-11 using the Maximum Likelihood (ML) method (1000 bootstrap replicates), and visualized with ITOL software. U-box proteins were classified into five distinct groups, as indicated by the different colors.

Figure 8

(A) Predicted cis-elements in NtU-box promoters. (B) Predicted cis-elements in SmU-box promoters. Different shapes and colors represent the different types of cis-elements. Annotations of cis-elements were listed in Supplementary Table S10 .

Figure 9

The expression patterns of tobacco and eggplant U-box genes in different tissues. (A) The expression patterns of NtU-box genes (B) The expression patterns of SmU-box genes. FPKM values for U-box genes were transformed by log2 (n+1).

Figure 10

The expression profiles of tobacco and eggplant U-box genes under abiotic stresses. (A) The expression patterns of NtU-box genes under drought stress. (B) The expression patterns of SmU-box genes under high temperature. FPKM values for U-box genes were transformed by log2 (n+1).

Figure 11

Disease symptoms of tobacco and eggplant seedlings. (A) Tobacco seedlings at 0 h and 96 h post-inoculation with Ras. (B) Eggplant seedlings at 0 h and 120 h post inoculation with Ras. The basal parts of stems were magnified and shown in the circles.

Figure 12

Expression profiles of U-box genes in response to Ras infection. (A) Relative expression level of 15 NtU-boxs in response to Ras infection. (B) Relative expression level of 8 SmU-boxs in response to Ras infection. Error bars are standard deviations of three biological replicates. Diverse letters on top of the bars indicate significant differences at P<0.05.